Antibiotic a 28829

ABSTRACT

PROCESS (AND PRODUCTS PRODUCED THEREBY) FOR MANUFACTURE OF A NEW ANTIBIOTIC, WHEREIN STREPTOMYCES ANTIBIOTICUS A 28829 IS CULTIVATED IN AN AQUEOUS NUTRIENT SOLUTION CONTAINING ASSIMILABLE SOURCES OF CARBON AND NITROGEN, AS WELL AS INORGANIC SALTS, UNDER AEROBIC CONDITIONS UNTIL SAID NUTRIENT SOLUTION DISPLAYS A SUBSTANTIAL ANTIBIOTIC ACTIVITY. THE ANTIBIOTIC OBTAINED IS EFFECTIVE AGAINST GRAM-POSITIVE MICROORGANISMS AND CERTAIN FUNGI.

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uolsslwsuell r7 Oct. 30, 1973 ANTIBIOTIC Filed oct. 4, 196e 2 Sheets-Sheet 2 Immo o.. o.- Qn o v o.m o w oN od United States Patent Ofce 3,769,418 VPatented Oct. 30, 1973 3,769,418 AN'IIBIOTIC A 28829 Vladimir Prelog, Zurich, Switzerland, Hans Zaehner,

Tubingen, Germany, and Hans Bickel, Binningen, Switzerland, assignors to Ciba-Geigy Corporation, Ardsley,

. FiledOct. 4, 1966, Ser'. No. 584,215 Claims priority, application Switzerland, Oct. 8, 1965, 13,926/6'5 Int. Cl. H61k 21/00 Cl. 424-121 3 Claims 'AiisTRACT on THE DISCLOSURE Process (and products produced thereby) for manufacture of a new antibiotic, wherein Streptomyces antibotcus A 28829 is cultivated in an aqueous nutrient solution containing assimilable sources of carbon and nitrogen, as well yas inorganic salts, under aerobic conditions until said nuftrient solution displays a substantial antibiotic activity.

The antibiotic obtained is effective against gram-positive microorganisms and certain fungi.

United States Department of Agriculture, Peoria, Ill.

The new strain Streptomyces antibotcus A 28829 forms a thin, profusely branched substrate mycelium from hyphae 0.6 to 0.8;; thick, from which air mycellium threads about 1p. broad emanate and from endogenic spores. The spores are ellipsoid, measure 0.6-4.4 X 0.5-

' 1.2/1. and have a smooth or at most slightly warty surface.

The sporechains are'monopodially branched and have rstraight or-wavy-lateral branches. When the strain is cultiyvated on a nutrient-containing `peptone, a melanod discoloration is observed.' The substrate mycelium is lightyellow, yellowish brown to light-brown, brownish grey or dark brown. The airmycelium isvelvety, initially chalky white or whitish grey and in the fully ripe state greyish brown to brownish grey or ash grey (cinereus). The strain grows under aerobic conditions'at temperatures from 18 vito 40, especially from 27 to 37 C. It is sensitive to lysozymes.v v

For further characterization of the strain its growth on a variety of nutrients is described below. The nutrients Nos. 5, 6, 7 and 10were prepared according to W. Lindenbein, Arch. Mikrobiol, 17, page 361 (1952); Nos.

2 and 4 according to Pridham et al., Antibiot, Ann. 1956/ l 57,' page 947'; No. 1 according to Gause et al., Problems yof Classification of Actinomycetes Antagonists (Russian), Nat. Verlag Medzig, Moscow, 1957,*page 22; No. 3 according to Tresner and Danger, J. Bact. 76, page 239 Y g. of meat extract (Lab-Lemco), 8.5 g: of sodium chloride, 17 g. ofv agar-agar and 1 litre of distilled waiter).

l (.1) Mineral substrate: Growththin, veil-like, light *brown- ,ish-grey; air mycelium velvety,` whitish grey to light grey;

. `substrate notcoloured .A

trogen source as well ,as inorganic salts under aerobic (1958), No` 5 according to he Manual of Methods of y"conditionsuntil the nutrient solution displays a substantial (2) Peptone-iron-agar: Growth wrinkly, whitish yellow,

no air mycelium; substrate not coloured (3) Yeast extract-agar: Growth thin, veil-like to wrinkly, brownish grey to dark brown; air mycelium velvety, brownish grey; substrate not coloured (4) Nitrate broth: Annular growth pimply, whitish yellow to light-yellow; air mycelium very sparse, forming a dusty coating, whitish grey; substrate deep yellow to light brown; strong nitrate reduction (5) Glucose-asparagineagar: Growth thin, veil-like, lightyellow to light brown; air mycelium sparse, velvety, whitish yellow to pale carmine or brownish grey; subtrate not coloured (6) Gelatine stab (27 C.): Surface growth punctiform,

light brown; air mycelium covered with our-like coating, whitish grey; substrate dark brown; liquefaction: 1 cm. after 5 days, 5 cm. after 10 days (7) Starch plate: Growth thin, veil-like, light brown; air mycelium covered with flour-like coating to velvety, brown grey; substrate not coloured; hydrolysis: 5 mm. after 10 days (8) Chromogen agar: Growth thin, veil-like, whitish yellow; air mycelium sparse, forming a dusty coating, whitish grey; melanine formation (9) Carvajals oatmeal agar: Growth thin, veil-like, whitish grey to brownish grey; substrate not coloured; air mycelium velvety, brownish grey (10) Litmus milk: Annular growth wrinkly, dark brown; air mycelium sparse, forming a dusty coating, whitish grey; substrate dark brown; peptonization, but little coagulation; pH=7.5 after 10 days.

The following carbon sources are used: d-glucose, L- arabinose, saccharose, d-Xylose, i-inositol, d-mannitol and d-fructose.

In its essential characteristics the strain 28829 corresponds to Streptomyces antzbotcus (Waksman et Woodruff), Waksman et Henrici 1948. In the following Table 1 the characteristic features of strain 28829 and of the type strain Streptomyces antbotcus strain IMRU 3435 are compared:

brownish grey. yellow, greenish grey.

Antibiotic AA 28829 is formed in cultivating yStreptomyces qntbotcus A 28829 or another strain having substantially the same characteristics. To produce antibiotic A 28829, Streptomyces antibotcus A 28829 or a micro'- organism that possesses its properties is grown in an aqueous nutrient solution containing a carbon and a niyantibiotic activity, whereupon antibiotic A 28829 is iso- '.'latedffrom it.

. correspondingto those ofantibi'otic A 28829 the test de- I TiIest micro-organisms for the presence of properties scribed'below with B otrytz's cnerea may be used.

The sources of carbon and nitrogen to be used in the cultix'i'lfationfmjay be," for example, glucose, saccharose, "fructose, stlir'ches,` mannitol, aminoacids (for example ,u'glycine), peptides, proteins'and their degradation prod- ,.:u'cts suchas peptornel orntryptone, meat extracts, water- 3. soluble constituents of cereal grains` such as maize or wheat, distillers solubles, cornsteep liquor, yeast, seeds (especially of the rape, soybean and cotton plant), ammonium salts and nitrates. Inorganic salts present in the nutrient solutionv may be, for example, chloride, carbonates, sulphates, nitrates or phosphates of alkali metals or alkaline earth metals, of magnesium, zinc, manganese or iron.

Cultivation is carried out under aerobic conditions, for example in a quiescent surface culture, or preferably submerged with shaking or stirring with air or oxygen in shaking bottles or in the known fermenters. A suitable temperature is within the -range from 27 to 37 C. In general, the nutrient solution displays a substantial antibacterial activityafter 2 to 5 days. It is of advantage to perform the cultivation in several stages, that is to sav at rst a pre-culture in a liquid nutrient medium is prepared and then used for inoculating the actual production medium, for example at the ratio of 1:20. The preculture may be prepared, for example, by transferring a spore mycelium formed by about 14 days growth on a solid nutrient medium to a liquid medium and allowing it to grow for 48 hours.

' The isolation of the antibiotic from the culture medium, that is to say from the mycelium, follows the usual practice, taking into consideration the chemical, physical and biological properties of the antibiotic. For testing the antibiotic activity in the individual isolation stagesas also in the culture medium-Botrytis cnerea is a particularly suitable test organism. The hyphae of this micro-organism are morphologically changed by antibiotic A 28829 (strong branching starting from a certain point so that structures resembling witches brooms result). The test is performed, for example, as a plate diffusion test in the following manner:

In the centre of the malt agar plates a circular area of 5 mm. diameter is inoculated with Botrytz's cinerea and the plates are incubated for 2 to 3 days at 24 C., during which the mycelium grows to a diameter of 2() to 30 mm. A solution of the substance to be tested is applied by means of round filter papers of 6 mm. diameter at a distance of about 5 mm. from the edge of the mycelium, whereupon the plates are incubated for 24 to 36 hours at 24 C. In the case of active solutions a morphological change of the hyphae can be observed up to a distance of 24 mm. from the round filter paper. Solutions having a concentration of 30'y/m1. of antibiotic A 28829 still form distinctly changed hyphae.

Antibiotic A 28829 possesses the following chemical and physical properties:

It is a lipophilic, neutral, colourless, crystalline substance which is soluble, interalia, in methanol, ethanol, acetone, ethyl acetate, chloroform, ether and petroleum ether. On crystallization from methanol it forms rodlets melting at 223 to 227 C. with decomposition.

Elementary analysis (percent): C=60.53, 60.79, 60.97; H- -8.52, 8.76, 8.32; N=1.89; B=1.35, 1.18; O (calcd.) :27.49.

The molecular weight* (determined by the thermoelectric 'method with methylenechloride as solvent) is ca. 868.

l The infrared spectrum (in potassium bromide solution) contains bands, inter alia, at 3420, 2915, 2880, 1735, 1630, 1515 (shoulder), 1165, 1370, 1328, 1287, 1263, 1222, 1202, 1178, 1127, 1100, 1070, 995, 923, 890 (shoulder.) 846, 795, 780, 757, 719 cm.-1 (see FIG. 1). f

The nuclear magnetic resonance spectrum is shown in FIG.2. l

The ultraviolet absorption spectrum contains no maximum between 210 and 400 ma.

In the thin-layer chromatogram on silica gel, eluant ethyl acetate, a single spot forms; Rf=0.75. Identification by spraying with concentrated sulphuric acid and heating to 140 C. or bioautographi'cally with Spiaria.

4 When the antibiotic is heated with dilute hydrochloric acid at C. for 30 minutes, no reducing sugar identifiable with ammoniacal silver nitrate or aniline hydrogen phthalate solution is formed.

Antibiotic A 28829 displays a good antibiotic activity towards Gram-positive micro-organisms, for example Bacillus subtilis, and towards fungi, for example Candida vulgaris, Paecilomyces variati and Spicarl'a. The following Table 2 shows the various concentrations at whichthe new antibiotic inhibits thsee micro-organisms in the plate diffusion test (6 mm. round llter papers impregnated with a solution of the antibiotic)..The figures given indicate the inhibiting zone for the antibiotic concentration concerned:

TABLE 2 Zonediameter in mm. at an antibioticconcentratlon ol- Further, the new antibiotic is distinguished by its activity against parasitic protozoae'especially plasmodiae and piroplasms, such as babesiae,t babesiellae and theileriae. It is also active against plasmodiae that have proved to be resistant towards known antimalaria medicaments. The new antibiotic can therefore be used pharmacologically on animals or as medicament, for example for the treatment of malaria, babesiasis, theileriosis, anaplasmosis and other infections. It can also be used as additive to animal fodder. Furthermore,it may be used as la disinfectant or preservative or forcombatting plant fungi.

The action against protozoa of the genera plasmodiae and babesiae has been demonstrated in the laboratory as follows: z

(la) Against Plasmodium berghei The action of the antibiotic has been tested in albino mice infected with a normally drug sensitive strain of P. berghei as well as in a strain rendered resistant to 7- chloro -'4 (4 diethylamino 1 methyl butylamino) quinoline-diphosphate. Mice are treated once daily orally with a solution of the product on the day of infection and each of the following three days..0ne day-after the end of treatment parasites are counted in a thin blood lm. Parasite densities in treated mice'are compared in Table 3 with those in untreated control mice. The ED50 is that dose which leads to a 50% suppression of parasitaemia in the treated mice as compared with control mice.

TABLE 3 Parasite No. density of mice (percent of Dose (mg/kg.) treated L, controls) Controls 30 r100 1 20 7516 3.-- w 20 42=k17 10 20 v0. OSO. 03

' (1b) vAgainst Plasmodima 'gallinaceu'm A similar test was made in'whichthe avian malaria P. gallz'naceum was used in infect 50 gchickspln this test the antibiotic was administered twice daily perI os as vstancesthat do not'react WithQ-the new compound, for

V groans 6 a suspension. Blood iilrns were examinedthe day after minutes at 125 C.). While stirring the culture at 700 to completing treatment. 'The resultisshown in Table 4. 900 revolutions per minute, it Vis incubated for 72 hours ,IRABLEQ' at 28 C. under a superatmospheric pressure of 1 atoms- Parasite phere (gauge) and while being aerated with 30 litres of N0 of density 5 air per minute. The mycelium is then removed from the gigs/g2g [day intljlcii (Dcerrllgf nutrient by vacuum filtration, and the antibiotic A 28829 is isolated from it by the method described below.

2 11% lgf (c) A culture incubated as described above under (b) 20 86 but only for 36 hours may be used for inoculating a larger O- fermenter (containing 300 litres of nutrient solution) at the volumetric ratio of 1:20, and the resulting culture may The'ED50is-interpo1`ated 38,744 Ing/kg then be used for inoculating a fermenter containing 3000 litres of nutrient solution, likewise at the volumetric ratio l(lo) Agalnst Bubesa rodha'z'n of 1;20

' (d) 3200 litres of culture medium of pH 6.3 are mixed with 64 kg. of Hyo-Supercel and filtered. The clear filtrate is discarded. The lmoist mixture of mycelium and Hytlo-Supercel is stirred for 30 minutes with 2 640 Albino mice are infected with B. rodha'z'ni and the prod- 15 Vuct administered orally 4in solution as described above for P. berghe. Thev resultjis shown in Table 5.

i TABLE 5 litres of 80% aqueous acetone and then centrifuged. The

Parasite centrifugate 1650 litres) is cautiously freed from acetone,

. "No. density and the residue (550 litres) is mixed with 82.5 kg. of

Dose (mgl/kg) gfelaf (plgf sodium chloride. and extracted at pH 5.9 with ethyl acetate -fm) at the volumetric ratio of 3:1 in a countercurrent extrac- 2 15 89 tor. The aqueous residue is once more mixed with 82.5

tg 4g 25 kg. of sodium chloride and extracted in identical manner.'

The combined extracts (570 litres) are caustiously concentrated to 10 litres. The oily residue is dissolved in 50 litres of petroleum ether (boiling range 50 to 70 C.), and the petroleum ether solution is extracted with 8X 6 litres of 85% methanol. The methanolic extracts contain the whole of the antibiotically active substance. On evaporation to dryness there are obtained 640 g. of crude The ED50 is interpolated as '2.4il.7 ing/kg.

The toxicity of the antibiotic has been measured by determining the LD50 i.e. the dose that kills 50% of the animals. The LD50 of the antibiotic in solution when administered orally to the mouse daily for 4 consecutive days is in the order of 15 mg./ kg. The therapeutic index compares favourably with standard drugs which are known antlblotlc A 28829' EXAMPLE 2 to exert a comparable chemotherapeutic action.

In the light of the above tests the antibiotic may be The lmycelium obtained as described in Example 1(b) envisaged for the treatment of normal or drug-resistant is extracted with 3 1 litre of ethyl acetate, the combined malaria in man ata daily dose of 2-5 mg. or a fraction extracts are concentrated to 500 ml., and the concentrate of this dose. i t is agitated three times with dilute acetic acid, water, dilute Antibiotic A 28829 may be used as a medicament, 'for 40 sodium bicarbonate solution and water. The ethyl acetate example in the form of pharmaceutical preparations consolution is dried over sodium sulphate and evaporated to taining -it ,in conjunction or admixture with an organic dryness under vacuum. According to the test with Botrytzs or inorganic pharmaceutical excipient suitable for enteral cinerea the residue contains substantially all of the activity or parenteral.,administrationf Suitable excipients are subof the culture medium. 7.09 g. of the residue are subjected to a Craig distribution over 100 stages. The solvent system used for this operation contains per 3 litres example gelatin, lactose, starches, magnesium stearate,

vegetable oils, benzyl alcohols or other known medicinal of carbon tetrachloride 2.55 litres of methanol and 0.45 excipients. The pharmaceuticalpreparations may be, for litre of water. The substance is liuslied with 150 ml. of solexample, tablets,Ldragees,.powders or suppositories, or vent mixture into the rst 3 elements of the fully autoin liquid form solutions, suspensions or emulsions. They matic apparatus. The antibiotic becomes enriched in the .may be sterilizedand/.r.theylmay contain auxiliaries stages 10 to 22; these fractions are combined and evap- 'such as preserving, stabilizing, wetting or emulsifying orated to dryness in vacuo, to yield 2.39 g. of a solid, agents. They may..also .contain further therapeutically brownish foam which crystallizes extensively when taken useful substances. l fup in the ether.

The following. examples illustrate the invention. EXAMPLE 3 EXAMPLE 1 A solution of 20 g. of the crude antibotic in absolute chloroform is chromatographed on a column of 500 g. (a) Th Strain Streptomwces am'botwus A 28829 1S of silica gel. The antibiotic is eluted with about 2 litres cultivated at 28,0 C- Ongyeast ggf (composllon: 4 g of of ethyl acetate. On concentration to 200 ml., the eluate yeast extract Dlfco, 195e 0f @alf extract DlfCO, 4 g- 0f 60 yields 14.75 g. of colourless crystals. Another 1.95 g. are

dextrose, 20 g.lof Bactoagar Difco, distilled water to make Obtained from the mother 1iqu0rs 1 mrc)- A fter 14vd ays SPQfulatd myclium has On recrystallization from methanol the antibiotic melts formed which is suspendediinphysiologicalsodium chloat 223 to 227 C .With decomposition i ride solution.`This suspension-of the fsporulatedmycelium Elementary analysis (percent): C=`60.53' 60.79, 60.97; Vj--(sfrongiii: 0,5% by'voitimoyiswisod ioinoouiaro 1/2 litro ,65 H=8 52, 8 76, 8,52, N- --i.89; B=1 35, 1,18; 0 (calci):

ofv nutrient 'solution ini-'a'2-litre' Yconicallla'slccontaining ,27,4%

andZO'g. of mannitol. The suspension i's'ii'icubated'for 48 with methylenechlor'ide assolverit)V is ca. 868."

PafatllS at feV01U10I}SP Cf minute- 70 ethyl acetate, av-singleV spot forms; Rf=0.75. Identifica- (b) 1.5 litres of a pre-culture prepared as described tionbysprayingmfwith kconcentrated sulphuricacidand 1 above are usedftoA in c t 1 late,i04 litres 0f a nutrientsolutiOn heating to` 140 C. or bi'autographicallywith Spicaria iii ofthe same composition asused f or making the pre-cul- .the followingmanner:u i i s 'l Y .ture; it is contained .ingu ,SQ-litreffermenter, and SPH u A-filter paper isplaced on an agar plate` inoeluatedwith value h'as beenadjustedtQfLS'priep to sterilization( fo r;20' 75. .Spicaria-germs and on top` the developed'tliinlayer' plate 7. is placed. After 15 minutes a suicient of antibiotic has diffused into the agar to cause the growth to be inhibited. The thin-layer plate and the filter paper are removed, and the agar plate is incubated for 20 hours at 36 C.

A solution of 20 mg. of antibiotic A 28829 in `2 ml. of dioxane is mixed with 2 m1. of N-hydrochloric acid and the whole is heated for 1/2 hour on a boiling water bath. The solution is neutralized with solid barium carbonate, evaporated to dryness and the residue is subjected to paper-chromatographic examination with the eluant n-butanol-i-glacial acetic acid-l-water (4:1:1). Reducing sugars can be identified neither With ammoniacal silver nitrate nor with aniline hydrogenphthalate solution.

What is claimed is:

1. Antibiotic A 28829, a lipophilic, neutral, colorless substance, soluble in methanol, ethanol, acetone, ethylacetate, chloroform, ether and petroleum ether, melting at 223-227 @dem when crystallized from methanol in rodlet form, having the elementary analysis C=60.76%; H=8.53%; N=1.89%; B=1.27 and 0:27.49, having a molecular weight about 868 and whose IR spectrum 8, (in KBr solution) contains bands at 3420, 2915, 2880, 1735, 1630, 1515 (shoulder), 1465, 1370, 1328, 1287, 1263, 1222, 1202, 1178, 1127, 1100, 1070, 995, 923, 890 (shoulder), 846, 795, 780, 757, 719 crm-1.

2. A process for producing antibiotic A 28829 which comprises cultivating Streptomyces antibioticus A 28829 NRRL 3207, in an aqueous nutrient medium containing assimilable sources of carbon,l nitrogen and inorganic salts, under aerobic conditions until a substantial quantity of said antibiotic is produced in said medium and then isolated from the mycelium.

3. A process as claimed in claim l2, wherein the antibiotic is crystallized from methanol.

No references Cited.

JEROME D. GOLDBERG, Primary Examiner U.S. C1. X.R. 195-80 

